Project Summary Vector-neutralizing immunity represents a major impediment to the induction of cellular immune responses using microbial-based vaccines. An ongoing trial of a novel recombinant Listeria monocytogenes (Lm) ?based cancer vaccine at our institution has yielded poor tumor antigen-specific immune responses. This result is consistent with other clinical trials with Lm-based vaccines. Importantly, we observed the marked expansion of ?? T cells in every subject. This finding is in direct contrast to murine models, where Lm-based vaccines generate a strong anti-tumor ?? T cell response to both endogenous Lm antigens and the tumor antigen. A particular invariant ?? T cell population is responsible for this effect and the target has been identified ? human V?9V?2 T cells respond to (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate of the MEP (or non-mevalonate) isoprenoid synthesis pathway. We hypothesize that HMBPP-deficient Lm does not activate the dominant human ?? T cell response and is a superior vaccine platform to generate ?? T cell responses for human immunotherapy when compared to vectors currently in clinical development. We propose that these ?? T cells facilitate the rapid clearance of Lm during 2 immunization, minimizing inflammation and the quantity and quality of antigen presentation to ?? T cells. The specific aims of this study are: 1: Test the hypothesis that rapid ?? T cell clearance of Listeria monocytogenes infected antigen presenting cells prevents generation of a ?? T cell response; and 2: Test the hypothesis that HMBPP-deficient Listeria monocytogenes is superior and safe vaccine for ?? T cell responses and does not generate ?? T cell responses. Our study design incorporates murine and humanized mouse models to determine whether a dominant ?? T cell response suppresses the capacity of Lm to generate ?? T cell responses and to evaluate comparative activation of ?? and ?? T cell responses in HMBPP-deficient versus standard Lm vaccines. This work is a necessary precursor to full trials of the vaccine in patients.